Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.

Local delivery of stem cell spheroids with protein/polyphenol self-assembling armor to improve myocardial infarction treatment via immunoprotection and immunoregulation.

Stem cell therapies have shown great potential for treating myocardial infarction (MI) but are limited by low cell survival and compromised functionality due to the harsh microenvironment at the disease site. Here, we presented a Mesenchymal stem cell (MSC) spheroid-based strategy for MI treatment by introducing a protein/polyphenol self-assembling armor coating on the surface of cell spheroids, which showed significantly enhanced therapeutic efficacy by actively manipulating the hostile pathological MI microenvironment and enabling versatile functionality, including protecting the donor cells from host immune clearance, remodeling the ROS microenvironment and stimulating MSC's pro-healing paracrine secretion. The underlying mechanism was elucidated, wherein the armor protected to prolong MSCs residence at MI site, and triggered paracrine stimulation of MSCs towards immunoregulation and angiogenesis through inducing hypoxia to provoke glycolysis in stem cells. Furthermore, local delivery of coated MSC spheroids in MI rat significantly alleviated local inflammation and subsequent fibrosis via mediation macrophage polarization towards pro-healing M2 phenotype and improved cardiac function. In general, this study provided critical insight into the enhanced therapeutic efficacy of stem cell spheroids coated with a multifunctional armor. It potentially opens up a new avenue for designing immunomodulatory treatment for MI via stem cell therapy empowered by functional biomaterials.

Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system, a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Macrophage infiltration in 3D cancer spheroids to recapitulate the TME and unveil interactions within cancer cells and macrophages to modulate chemotherapeutic drug efficacy.

BACKGROUND: Recapitulating the tumor microenvironment (TME) in vitro remains a major hurdle in cancer research. In recent years, there have been significant strides in this area, particularly with the emergence of 3D spheroids as a model system for drug screening and therapeutics development for solid tumors. However, incorporating macrophages into these spheroid cultures poses specific challenges due to the intricate interactions between macrophages and cancer cells. METHODS: To address this issue, in this study, we established a reproducible healthy multicellular 3D spheroid culture with macrophage infiltrates in order to mimic the TME and modulate the drug's efficacy on cancer cells in the presence of macrophages. A 3D spheroid was established using the human cancer cell line CAL33 and THP1 cell derived M0 macrophages were used as a source of macrophages. Cellular parameters including tumour metabolism, health, and mitochondrial mass were analysed in order to establish ideal conditions. To modulate the interaction of cancer cells with macrophage the ROS, NO, and H2O2 levels, in addition to M1 and M2 macrophage phenotypic markers, were analyzed. To understand the crosstalk between cancer cells and macrophages for ECM degradation, HSP70, HIF1α and cysteine proteases were examined in spheroids using western blotting and qPCR. RESULTS: The spheroids with macrophage infiltrates exhibited key features of solid tumors, including cellular heterogeneity, metabolic changes, nutrient gradients, ROS emission, and the interplay between HIF1α and HSP70 for upregulation of ECM degradading enzymes. Our results demonstrate that tumor cells exhibit a metabolic shift in the presence of macrophages. Additionally, we have observed a shift in the polarity of M0 macrophages towards tumor-associated macrophages (TAMs) in response to cancer cells in spheroids. Results also demonstrate the involvement of macrophages in regulating HIF-1α, HSP70, and ECM degradation cysteine proteases enzymes. CONCLUSIONS: This study has significant implications for cancer therapy as it sheds light on the intricate interaction between tumor cells and their surrounding macrophages. Additionally, our 3D spheroid model can aid in drug screening and enhance the predictive accuracy of preclinical studies. The strength of our study lies in the comprehensive characterization of the multicellular 3D spheroid model, which closely mimics the TME.

Formation and Growth of Co-Culture Tumour Spheroids: New Compartment-Based Mathematical Models and Experiments.

Co-culture tumour spheroid experiments are routinely performed to investigate cancer progression and test anti-cancer therapies. Therefore, methods to quantitatively characterise and interpret co-culture spheroid growth are of great interest. However, co-culture spheroid growth is complex. Multiple biological processes occur on overlapping timescales and different cell types within the spheroid may have different characteristics, such as differing proliferation rates or responses to nutrient availability. At present there is no standard, widely-accepted mathematical model of such complex spatio-temporal growth processes. Typical approaches to analyse these experiments focus on the late-time temporal evolution of spheroid size and overlook early-time spheroid formation, spheroid structure and geometry. Here, using a range of ordinary differential equation-based mathematical models and parameter estimation, we interpret new co-culture experimental data. We provide new biological insights about spheroid formation, growth, and structure. As part of this analysis we connect Greenspan's seminal mathematical model to co-culture data for the first time. Furthermore, we generalise a class of compartment-based spheroid mathematical models that have previously been restricted to one population so they can be applied to multiple populations. As special cases of the general model, we explore multiple natural two population extensions to Greenspan's seminal model and reveal biological mechanisms that can describe the internal dynamics of growing co-culture spheroids and those that cannot. This mathematical and statistical modelling-based framework is well-suited to analyse spheroids grown with multiple different cell types and the new class of mathematical models provide opportunities for further mathematical and biological insights.

Exploring the interaction between extracellular matrix components in a 3D organoid disease model to replicate the pathophysiology of breast cancer.

In vitro models are necessary to study the pathophysiology of the disease and the development of effective, tailored treatment methods owing to the complexity and heterogeneity of breast cancer and the large population affected by it. The cellular connections and tumor microenvironments observed in vivo are often not recapitulated in conventional two-dimensional (2D) cell cultures. Therefore, developing 3D in vitro models that mimic the complex architecture and physiological circumstances of breast tumors is crucial for advancing our understanding of the illness. A 3D scaffold-free in vitro disease model mimics breast cancer pathophysiology by allowing cells to self-assemble/pattern into 3D structures, in contrast with other 3D models that rely on artificial scaffolds. It is possible that this model, whether applied to breast tumors using patient-derived primary cells (fibroblasts, endothelial cells, and cancer cells), can accurately replicate the observed heterogeneity. The complicated interactions between different cell types are modelled by integrating critical components of the tumor microenvironment, such as the extracellular matrix, vascular endothelial cells, and tumor growth factors. Tissue interactions, immune cell infiltration, and the effects of the milieu on drug resistance can be studied using this scaffold-free 3D model. The scaffold-free 3D in vitro disease model for mimicking tumor pathophysiology in breast cancer is a useful tool for studying the molecular basis of the disease, identifying new therapeutic targets, and evaluating treatment modalities. It provides a more physiologically appropriate high-throughput platform for screening large compound library in a 96-384 well format. We critically discussed the rapid development of personalized treatment strategies and accelerated drug screening platforms to close the gap between traditional 2D cell culture and in vivo investigations.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

An Integrative Bioorthogonal Nanoengineering Strategy for Dynamically Constructing Heterogenous Tumor Spheroids.

Although tumor models have revolutionized perspectives on cancer aetiology and treatment, current cell culture methods remain challenges in constructing organotypic tumor with in vivo-like complexity, especially native characteristics, leading to unpredictable results for in vivo responses. Herein, the bioorthogonal nanoengineering strategy (BONE) for building photothermal dynamic tumor spheroids is developed. In this process, biosynthetic machinery incorporated bioorthogonal azide reporters into cell surface glycoconjugates, followed by reacting with multivalent click ligand (ClickRod) that is composed of hyaluronic acid-functionalized gold nanorod carrying dibenzocyclooctyne moieties, resulting in rapid construction of tumor spheroids. BONE can effectively assemble different cancer cells and immune cells together to construct heterogenous tumor spheroids is identified. Particularly, ClickRod exhibited favorable photothermal activity, which precisely promoted cell activity and shaped physiological microenvironment, leading to formation of dynamic features of original tumor, such as heterogeneous cell population and pluripotency, different maturation levels, and physiological gradients. Importantly, BONE not only offered a promising platform for investigating tumorigenesis and therapeutic response, but also improved establishment of subcutaneous xenograft model under mild photo-stimulation, thereby significantly advancing cancer research. Therefore, the first bioorthogonal nanoengineering strategy for developing dynamic tumor models, which have the potential for bridging gaps between in vitro and in vivo research is presented.

Spheroid on-demand printing and drug screening of endothelialized hepatocellular carcinoma model at different stages.

Hepatocellular carcinoma (HCC) poses a significant threat to human health and medical care. Its dynamic microenvironment and stages of development will influence the treatment strategies in clinics. Reconstructing tumor-microvascular interactions in different stages of the microenvironment is an urgent need forin vitrotumor pathology research and drug screening. However, the absence of tumor aggregates with paracancerous microvascular and staged tumor-endothelium interactions leads to bias in the antitumor drug responses. Herein, a spheroid-on-demand manipulation strategy was developed to construct staged endothelialized HCC models for drug screening. Pre-assembled HepG2 spheroids were directly printed by alternating viscous and inertial force jetting with high cell viability and integrity. A semi-open microfluidic chip was also designed to form a microvascular connections with high density, narrow diameter, and curved morphologies. According to the single or multiple lesions in stages Ⅰ or Ⅰ HCC, endothelialized HCC models from micrometer to millimeter scale with dense tumor cell aggregation and paracancerous endothelial distribution were successively constructed. A migrating stage Ⅰ HCC model was further constructed under TGF-ßtreatment, where the spheroids exhibited a more mesenchymal phenotype with a loose cell connection and spheroid dispersion. Finally, the stage ⅠHCC model showed stronger drug resistance compared to the stage Ⅰ model, while the stage III showed a more rapid response. The corresponding work provides a widely applicable method for the reproduction of tumor-microvascular interactions at different stages and holds great promise for the study of tumor migration, tumor-stromal cell interactions, and the development of anti-tumor therapeutic strategies.

In Situ Spatial Analysis of Metabolic Heterogeneity in Single Living Tumor Spheroids Using Nanocapillary-Based Electrospray Ionization Mass Spectroscopy.

Spatial metabolomic analysis of individual tumor spheroids can help investigate metabolic rearrangements in different cellular regions of a spheroid. In this work, a nanocapillary-based electrospray ionization mass spectroscopy (ESI-MS) method is established that could realize the spatial sampling of cellular components in different regions of a single living tumor spheroid and the subsequent MS analysis for a metabolic study. During the penetration of the nanocapillary into the spheroid for sampling, this "wound surface" at the outer layer of the spheroid takes only 0.1% of the whole area that maximally maintains the cellular activity inside the spheroid for the metabolic analysis. Using the ESI-MS analysis, different metabolic activities in the inner and outer (upper and lower) layers of a single spheroid are revealed, giving a full investigation of the metabolic heterogeneity inside one living tumor spheroid for the first time. In addition, the metabolic activities between the outer layer of the spheroid and two-dimensional (2D)-cultured cells show obvious differences, which suggests more frequent cell-cell and cell-extracellular environment interactions during the culture of the spheroid. This observation not only establishes a powerful tool for the in situ spatial analysis of the metabolic heterogeneity in single living tumor spheroids but also provides molecular information to elucidate the metabolic heterogeneity in this three-dimensional (3D)-cultured cell model.

Scaffold-based spheroid models of glioblastoma multiforme and its use in drug screening.

Among several types of brain cancers, glioblastoma multiforme (GBM) is a terminal and aggressive disease with a median survival of 15 months despite the most intensive surgery and chemotherapy. Preclinical models that accurately reproduce the tumor microenvironment are vital for developing new therapeutic alternatives. Understanding the complicated interactions between cells and their surroundings is essential to comprehend the tumor's microenvironment, however the monolayer cell culture approach falls short. Numerous approaches are used to develop GBM cells into tumor spheroids, while scaffold-based spheroids provides the opportunity to investigate the synergies between cells as well as cells and the matrix. This review summarizes the development of various scaffold-based GBM spheroid models and the prospective for their use as drug testing systems.

Three-Dimensional Spheroids for Cancer Research.

In vitro cell culture is one of the most widely used tools used today for increasing our understanding of various things such as protein production, mechanisms of drug action, tissue engineering, and overall cellular biology. For the past decades, however, cancer researchers have relied heavily on conventional two-dimensional (2D) monolayer culture techniques to test a variety of aspects of cancer research ranging from the cytotoxic effects of antitumor drugs to the toxicity of diagnostic dyes and contact tracers. However, many promising cancer therapies have either weak or no efficacy in real-life conditions, therefore delaying or stopping altogether their translating to the clinic. This is, in part, due to the reductionist 2D cultures used to test these materials, which lack appropriate cell-cell contacts, have altered signaling, do not represent the natural tumor microenvironment, and have different drug responses, due to their reduced malignant phenotype when compared to real in vivo tumors. With the most recent advances, cancer research has moved into 3D biological investigation. Three-dimensional (3D) cultures of cancer cells not only recapitulate the in vivo environment better than their 2D counterparts, but they have, in recent years, emerged as a relatively low-cost and scientifically accurate methodology for studying cancer. In this chapter, we highlight the importance of 3D culture, specifically 3D spheroid culture, reviewing some key methodologies for forming 3D spheroids, discussing the experimental tools that can be used in conjunction with 3D spheroids and finally their applications in cancer research.

Cell Viability Assay with 3D Prostate Tumor Spheroids.

WST-8 (Cell Counting Kit 8; CCK-8) is the last generation tetrazolium-based cell viability assay and has recently been accepted as a validated method for measuring the cell viability of 3D in vitro models. Here, we describe how to form 3D prostate tumor spheroids using the polyHEMA technique, apply drug treatments and WST-8 assay to these spheroids, and calculate their cell viability. The advantages of our protocol are the formation of spheroids without adding extracellular matrix components, and the elimination of the critique handling process needed for transferring spheroids. Although this protocol exemplifies the determination of percentage cell viability in PC-3 prostate tumor spheroids, it can be adapted and optimized for other prostate cell lines and other types of cancers.

Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

A mechanobiological model for tumor spheroid evolution with application to glioblastoma: A continuum multiphysics approach.

BACKGROUND: Spheroids are in vitro quasi-spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. The main objective of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. METHOD: We propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroid evolution under in vitro suspension, namely, nutrient diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. RESULTS: Our model is able to explain in a novel way quite different setups, such as spheroid growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. CONCLUSIONS: Glioblastoma tumor spheroid evolution is driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease.

Combining preclinical tools and models to unravel tumor complexity: Jump into the next dimension.

Tumors are complex and heterogeneous diseases characterized by an intricate milieu and dynamically in connection with surrounding and distant tissues. In the last decades, great efforts have been made to develop novel preclinical models able to recapitulate the original features of tumors. However, the development of an in vitro functional and realistic tumor organ is still utopic and represents one of the major challenges to reproduce the architecture of the tumor ecosystem. A strategy to decrypt the whole picture and predict its behavior could be started from the validation of simplified biomimetic systems and then proceed with their integration. Variables such as the cellular and acellular composition of tumor microenvironment (TME) and its spatio-temporal distribution have to be considered in order to respect the dynamic evolution of the oncologic disease. In this perspective, we aim to explore the currently available strategies to improve and integrate in vitro and in vivo models, such as three-dimensional (3D) cultures, organoids, and zebrafish, in order to better understand the disease biology and improve the therapeutic approaches.

3D Tumor Models in Urology.

Three-dimensional tumor models have become established in both basic and clinical research. As multicellular systems consisting of tumor and tumor-associated cells, they can better represent tumor characteristics than monocellular 2D cultures. In this review, we highlight the potential applications of tumor spheroids and organoids in the field of urology. Further, we illustrate the generation and characteristics of standardized organoids as well as membrane-based 3D in vitro models in bladder cancer research. We discuss the technical aspects and review the initial successes of molecular analyses in the three major urologic tumor entities: urinary bladder carcinoma (BCa), prostate carcinoma (PCa), and renal cell carcinoma (RCC).

Generation of 3D Tumor Spheroids for Drug Evaluation Studies.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.